1. Field of the Invention
The invention relates generally to the fields of immunology, microbiology and inflammatory bowel disease and more specifically to the diagnosis and treatment of inflammatory bowel disease using Pseudomonas antigens.
2. Background Information
Inflammatory bowel disease (IBD) is the collective term used to describe two gastrointestinal disorders of unknown etiology: Crohn""s disease (CD) and ulcerative colitis (UC). The course and prognosis of IBD, which occurs world-wide and is reported to afflict as many as two million people, varies widely. Onset of IBD is predominantly in young adulthood with diarrhea, abdominal pain, and fever the three most common presenting symptoms. The diarrhea may range from mild to severe, and anemia and weight loss are additional common signs of IBD. Ten percent to fifteen percent of all patients with IBD will require surgery over a ten year period. In addition, patients with IBD are at increased risk for the development of intestinal cancer. Reports of an increased occurrence of psychological problems, including anxiety and depression, are perhaps not surprising symptoms of what is often a debilitating disease that strikes people in the prime of life.
Unfortunately, the available therapies for inflammatory bowel disease are few, and both diagnosis and treatment have been hampered by a lack of knowledge regarding the etiology of the disease. What is clear, however, is that a combination of genetic factors, exogenous triggers and endogenous microflora can contribute to the immune-mediated damage to the intestinal mucosa seen in inflammatory bowel disease. In Crohn""s disease, bacteria have been implicated in initiation and progression of the disease: the intestinal inflammation in Crohn""s disease is notable for its frequent responsiveness to antibiotics and susceptibility to bacterial fecal flow. Common intestinal colonists and novel pathogens have been implicated in Crohn""s disease by direct detection or by disease associated anti-microbial immune responses. Furthermore, in many genetically susceptible animal models of chronic colitis, lumenal micro-organisms are a necessary cofactor for disease; animals housed in a germ-free environment do not develop colitis. However, despite much direct and indirect evidence for a role for enteric microorganisms in Crohn""s disease, the pathogenic organisms contributing to the immune dysregulation seen in this disease have not been identified.
Identification of the involved microbial species would provide a basis for the discovery of new antibiotics and other drugs for treating Crohn""s disease, such drugs ameliorating disease by eliminating the microbial inducers of disease. Furthermore, identification of the involved microbial species would provide a basis for novel vaccines and for diagnosing Crohn""s disease based on the presence of reactivity in patient sera against the identified microbial organisms.
Thus, there is a need for identification of the microbial species that play a role in inflammatory bowel disease such as Crohn""s disease. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides a method of identifying an agent useful in treating Crohn""s disease. This method is practiced by culturing P. fluorescens under conditions that support growth; contacting the P. fluorescens with an agent; and assaying for reduced growth or viability of the P. fluorescens as compared to the growth or viability in the absence of the agent, where the reduced growth or viability of the P. fluorescens indicates that the agent is an anti-P. fluorescens agent useful in treating Crohn""s disease. Such an agent can be, for example, an antibiotic. An effective amount of an anti-P. fluorescens agent identified according to a method of the invention can be administered to an individual to prevent or treat Crohn""s disease. The anti-P. fluorescens agent identified and administered can be, for example, an antibiotic or a combination of two or more antibiotics.
The invention also provides a method of preventing or treating Crohn""s disease in an individual by administering to the individual an effective amount of an anti-Pseudomonas agent. Such an anti-Pseudomonas agent can be, for example, an anti-P. fluorescens agent. If desired, an anti-Pseudomonas agent can be administered in a manner which is optimized for effectivity against P. fluorescens. Anti-Pseudomonas agents useful for preventing or treating Crohn""s disease include antibiotics such as xcex2-lactamase-resistant penicillin formulations, aminoglycosides and fluoroquinolones. In one embodiment, the anti-Pseudomonas agent administered to prevent or treat Crohn""s disease is the antibiotic vancomycin. If desired, two or more antibiotics can be administered in combination to prevent or treat Crohn""s disease according to a method of the invention.
Further provided by the invention is a method of preventing or treating Crohn""s disease in an individual by administering to the individual an effective dose of an anti-Pseudomonas vaccine. Such a vaccine can be, for example, an anti-P. fluorescens vaccine. Anti-Pseudomonas vaccines useful in the invention include killed whole Pseudomonas such as killed whole P. fluorescens. 
In one embodiment, the anti-Pseudomonas vaccine is a purified antigen. Thus, the invention additionally provides a method of preventing or treating Crohn""s disease in an individual by administering to the individual an effective dose of a purified Pseudomonas antigen, or a tolerogenic fragment thereof. In a method of the invention, the purified Pseudomonas antigen can be, for example, a purified P. fluorescens antigen or a tolerogenic fragment thereof. The methods of the invention can be practiced with a purified P. fluorescens antigen such as pbrA, or a tolerogenic fragment thereof. In one embodiment, the methods of the invention are practiced with a purified pbrA antigen having the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 3, or a tolerogenic fragment of one of these sequences.
A purified Pseudomonas antigen, or a tolerogenic fragment thereof, useful in the invention also can be PFTR, or a tolerogenic fragment thereof, provided that the fragment is not I-2 or a fragment thereof. For example, the methods of the invention can be practiced with purified PFTR having the amino acid sequence SEQ ID NO: 5, or a tolerogenic fragment thereof. Additional Pseudomonas antigens useful in the invention include, but are not limited to, outer membrane proteins, toxins, lipopolysaccharide (LPS), exotoxin A and TonB and tolerogenic fragments thereof.
Further provided by the invention is a method of preventing or treating Crohn""s disease in an individual by administering to the individual an agent that reduces the expression or activity of pbrA, thereby reducing the growth or viability of P. fluorescens in the individual. In one embodiment, such an agent reduces the expression of pbrA. Exemplary agents that reduce the expression of pbrA include, without limitation, pbrA antisense nucleic acid molecules and sequence-specific ribonucleases.
The invention additionally provides a method of preventing or treating Crohn""s disease in an individual by administering to the individual an agent that reduces the expression or activity of PFTR, thereby reducing the growth or viability of P. fluorescens in the individual. Agents which reduce the expression of PFTR include PFTR antisense nucleic acid molecules and sequence-specific ribonucleases. Agents useful in the invention further include inhibitors of PFTR enzymatic function such as inhibitors of ferrodoxin activity, for example, competitive inhibitors of ferrodoxin activity.
The invention also provides a method of diagnosing Crohn""s disease in a individual by obtaining a sample from the individual; contacting the sample with pbrA, or an immunoreactive fragment thereof, under conditions suitable to form a complex of pbrA, or the immunoreactive fragment thereof, and antibody to pbrA; and detecting the presence or absence of the complex, where the presence of the complex indicates that the individual has Crohn""s disease. In such a diagnostic method of the invention, the presence or absence of the complex can be detected, for example, with a detectable secondary antibody. The methods of the invention can be practiced, for example, with pbrA having the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 3, or with an immunoreactive fragment of one of these sequences.
The invention also provides a method of diagnosing Crohn""s disease in a individual by obtaining a sample from the individual; contacting the sample with PFTR, or an immunoreactive fragment thereof, under conditions suitable to form a complex of PFTR, or the immunoreactive fragment thereof, and antibody to PFTR; and detecting the presence or absence of the complex, provided that the immunoreactive fragment is not I-2 or a fragment thereof, where the presence of the complex indicates that the individual has Crohn""s disease. In one embodiment, the presence or absence of the complex is detected with a detectable secondary antibody. In a further embodiment, the diagnostic method of the invention is practiced with PFTR having the amino acid sequence SEQ ID NO: 5, or an immunoreactive fragment thereof.